Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Western blot analysis results for ERS-associated proteins. (A) The representative blotting images and (B) quantified data demonstrate elevated p-PERK, eIF2a, CHOP, Bax/Bcl-2, Caspase-12 and cleaved Caspase-3 level in L539fs/47-hERG cells, which can be reversed by 4-PBA. Each experiment was repeated at least three times. # P<0.05 between the three model cell groups; * P<0.05 between two subgroups treated with or without 4-PBA intervention. P<0.05 was considered to indicate a statistically significant difference. ERS, endoplasmic reticulum stress; hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Western Blot

Journal: International Journal of Molecular Medicine

Article Title: Human ether-à-go-go-related gene mutation L539fs/47-hERG leads to cell apoptosis through the endoplasmic reticulum stress pathway

doi: 10.3892/ijmm.2019.4049

Figure Lengend Snippet: Proposed mechanisms of mutated hERG-induced cell apoptosis. Mutated L539fs/47-hERG protein retained in the ER lumen provokes ERS. Elevated expression levels of GRP78 activate the PERK-eIF2α-CHOP pathway, which is important in the mechanism of L539fs/47-hERG-induced ERS-mediated cell apoptosis. ER-specific caspase-12 is involved in the apoptotic mechanisms caused by the hERG mutation through cleaving and activating caspase-3. The pair of apoptosis factors Bax/Bcl-2 also regulate L539fs/47-hERG-induced cell apoptosis. hERG, human ether-à-go-go-related gene; WT, wild-type; 4PBA, 4-phenyl butyric acid; ER, endoplasmic reticulum; ERS, ER stress; ATF6, activating transcription factor 6; IRE1, inositol-requiring enzyme 1; p-PERK, phosphorylated protein kinase R-like endoplasmic reticulum kinase; eIF2a; eukaryotic translation-initiation factor-2α; CHOP, C/EBP homologous protein; Bcl2, B-cell lymphoma 2; Bax, Bcl-2-associated X protein.

Article Snippet: The primary antibodies were as follows: hERG (sc-377388; 1:500; Santa Cruz Biotechnology, Inc., Dallas, TX, USA), GRP78 (cat. no. WL0781; 1:1,000; Wanleibio Co., Ltd., Shanghai, China), phosphorylated (p-)PERK (cat. no. bs-23340R; 1:500; Bioss, Beijing, China), p-eIF2α (cat. no. 3398; 1:1,000; Cell Signaling Technology, Inc., Danvers, MA, USA), CHOP (cat. no. WL00880; 1:1,000), Bax (cat. no. WL01637; 1:500), Bcl-2 (cat. no. WL01556; 1:500), cleaved-caspase-3 (cat. no. WL01857; 1:500; all Wanleibio Co., Ltd.), caspase-12 (cat. no. 2202; 1:1,000) and GAPDH (cat. no. sc-25778; 1:1,000; both Santa Cruz Biotechnology, Inc.).

Techniques: Expressing, Mutagenesis

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 4. FGF21 alleviates the hypoxia‑induced ERS by modulating the expression of ERS‑related proteins in HPAECs. HPAECs were treated as indicated and protein expression levels were examined by western blotting. Representative blots and quantification are shown for (A) BiP, (B) p‑PERK and PERK, (C) the ratio of p‑PERK/PERK, (D) CHOP, (E) Bcl‑2. (F) Relative expression of the ERS‑dependent apoptotic protein caspase‑4 in HPAECs was analysed by western blotting. GAPDH was used as an internal control. All experiments were performed in triplicate, and data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. N group; ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. FGF21, fibroblast growth factor 21; ERS, endoplasmic reticulum stress; HPAECs, human pulmonary arterial endothelial cells; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcrip- tion factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Expressing, Western Blot, Control, Standard Deviation, Binding Assay

Journal: International journal of molecular medicine

Article Title: FGF21 attenuates hypoxia‑induced dysfunction and apoptosis in HPAECs through alleviating endoplasmic reticulum stress.

doi: 10.3892/ijmm.2018.3705

Figure Lengend Snippet: Figure 6. Effect of FGF21 on NO and ET‑1 secretion in HPAECs. (A) HPAECs were treated as indicated and at the end of treatment, the cell culture medium was collected and assayed by ELISA for the levels of secreted NO and (B) the levels of secreted ET‑1. Data are expressed as the mean ± standard deviation (n=3). **P<0.01 vs. the N group; #P<0.05 and ##P<0.01 vs. the H group; &&P<0.01 vs. the H+T group. (C) Schematic of the proposed mechanism by which FGF21 attenuates hypoxia‑induced apoptosis and dysfunction by alleviating ERS in HPAECs. FGF21, fibroblast growth factor 21; NO, nitric oxide; ET‑1, endothelin‑1; HPAECs, human pulmonary arterial endothelial cells; ERS, endoplasmic reticulum stress; N, normoxia; H, hypoxia; F, FGF21; S, salubrinal; T, tunicamycin; BiP, binding immunoglobulin protein; p‑, phosphorylated; PERK, protein kinase R‑like endoplasmic reticulum kinase; CHOP, transcription factor C/EBP homologous protein; Bcl‑2, B cell lymphoma-2.

Article Snippet: Rabbit antibodies against phosphorylated (p-) PERK were purchased from Biorbyt (cambridge, UK).

Techniques: Cell Culture, Enzyme-linked Immunosorbent Assay, Standard Deviation, Binding Assay